【作者】 陈龙正；

【导师】 陈劲枫；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2008， 博士

【摘要】 多倍化是高等植物进化过程的重要阶段,是植物进化的主要动力之一。研究表明,异源多倍体在形成的早期会出现广泛的基因组遗传和表观遗传变化,这些变化直接关系到物种的形成和稳定。然而,现存的天然多倍体大多形成于成千上万年前,很难确定进化中种种不稳定现象发生的具体过程和机制。虽然分子标记及比较基因组学的发展为认识杂交及多倍化进程提供了重要依据,但由于分子进化机制的复杂性,这方面的相关机理知之甚少。人工合成的异源多倍体则为这方面的研究提供了良好的模式系统。通过这种模式系统,可以精确比较亲本二倍体种与人工异源多倍体早期世代间的基因组变化特点,从而为丰富多倍体物种进化理论提供重要的实证。本研究基于实验室已合成的甜瓜属远缘杂种F₁及人工合成的异源双二倍体及其不同自交世代,比较研究了杂种F₁及异源双二倍体的减数分裂染色体行为特征、基因组位点变化和甲基化模式变化特点,探索了远缘杂交与多倍化引发远缘杂种F₁和异源双二倍体基因组发生遗传和表观遗传变化的机制。具体如下:1.远缘杂种F₁及异源双二倍体减数分裂染色体行为与花粉育性研究研究了野生种C.hystrix（2n=2X=24）与栽培黄瓜C.sativus L.（2n=2X=14）远缘杂种F₁（2n=2X=19）、双二倍体及双二倍体（2n=4X=38）自交1～4代的减数分裂染色体行为特征与花粉育性。结果表明:减数分裂中期Ⅰ,杂种F₁花粉母细胞（pollen mother cell,PMC）染色体主要以单价体存在,染色体构型为:16.75Ⅰ+0.5Ⅱ+0.25Ⅲ+0.13Ⅳ;双二倍体染色体构型以二价体为主,伴随着自交,平均每个PMC所含二价体比例逐渐增加,非二价体配对比例减少。四分体时期,F₁不能形成正常的四分孢子,主要以多分孢子形态存在;双二倍体以四分孢子为主,并随着自交世代的增高,其比例增加。花粉育性观察发现,随着自交世代的增高,双二倍体花粉育性呈增加趋势。以上结果暗示双二倍体在遗传上逐渐趋于稳定。双二倍体PMCs在后期Ⅰ、Ⅱ普遍含有落后染色体、染色体桥及染色体不均等分离等异常现象,这可能是导致其花粉育性低于期望值的原因。2.甜瓜属远缘杂种F₁及人工异源双二倍体早期基因组序列变化研究利用12对Eco RI/Mse I选择性引物,对甜瓜属人工异源双二倍体C.hytivus及其二倍体父母本进行AFLP分析,结果发现多倍体基因组在形成早期发生了广泛的序列变化。基因组序列变化始于F₁代,变化最剧烈的时期发生在S₀到S₁代。但在S₁到S₂代基因组序列变化比率基本接近,暗示了伴随着异源双二倍体的自交过程,早期基因组表现出稳定的趋势,说明基因组序列变化是一个快速的事件。基因组序列变化主要表现为部分亲本片段丢失、新片段产生以及后代对亲本某些位点的跳跃式继承。对变化的序列进行深入研究发现,变化的序列主要是重复序列。实验还观察到少部分双亲差异性条带呈现出偏父性遗传的特点。不同分类群的异源多倍体在二倍化过程中,正反交序列丢失往往表现出不同特征,暗示了在不同物种中核质互作在多倍体进化过程的作用不同。为了研究细胞质背景对基因组序列丢失的影响,利用23对Eco RI-NN/Mse I-NNN选择性引物对Cucumishystrix与栽培黄瓜C.sativus的正反交F₁、异源双二倍体及亲本DNA进行AFLP分析。结果表明:杂种F₁与异源双二倍体基因组发生了广泛的序列丢失。正交后代与反交后代在丢失亲本基因组序列的频率上差异不显著,并且在序列丢失时间（均始于F₁代）及丢失类型上也表现出相同的特点,表明核质互作并不是影响序列丢失的主要因素。实验还发现,不论正交还是反交,后代丢失普通黄瓜基因组序列数大于丢失野生种基因组序列数,表明甜瓜属多倍化早期阶段易于丢失染色体数比较少的亲本基因组序列。3.甜瓜属远缘杂交及多倍化诱发基因组DNA胞嘧啶甲基化变化特征研究为了研究DNA甲基化在甜瓜属人工异源多倍体形成过程中的作用,利用methylation-sensitive amplification polymorphism（MSAP）技术,对甜瓜属远缘杂种F₁、人工异源双二倍体及亲本基因组DNA进行研究。结果发现,远缘杂种F₁及人工异源双二倍体基因组发生了胞嘧啶甲基化模式变化,表明远缘杂交及多倍化均可诱发基因组胞嘧啶甲基化发生变化。甲基化变化根据变化模式的不同可以分为五种类型,主要是脱甲基化类型（甲基化水平降低）和过甲基化类型（甲基化水平升高）,并且发生过甲基化变化的位点数是发生脱甲基化变化的2-4倍,说明甲基化变化主要表现为亲本基因组的部分位点在后代中发生过甲基化。研究还发现,与父本基因组相比,母本基因组在远缘杂交后代中易发生过甲基化变化,暗示了DNA甲基化在识别和保护自身DNA中起重要作用。对异源双二倍体不同自交世代甲基化进行研究,在S₁和S₂代之间发生甲基化模式变化的位点数显著多于S₂和S₃代,表明伴随着自交世代的升高,双二倍体基因组胞嘧啶甲基化变化剧烈程度逐渐降低,基因组逐渐趋于稳定,DNA甲基化可能在遗传二倍化过程中起重要作用。为了研究甜瓜属异源双二倍体在形成及稳定过程中甲基化变化位点的特征特性,我们对远缘杂交及多倍化诱发的基因组胞嘧啶甲基化变化位点进行分析。结果发现,在克隆测序的15条序列中,有7条序列与已知功能基因或调控基因具有较高的相似性。根据测序结果设计特异引物,进行PCR验证分析,发现MSAP多态性由DNA甲基化修饰引起。并且RT-PCR分析发现胞嘧啶甲基化可能参与了甜瓜属人工异源双二倍体基因表达的调控,表明其在异源多倍体二倍体化过程中起到重要作用。Southernblot结果发现,甲基化变化序列不仅有单拷贝序列,而且也有部分是多拷贝序列。为了研究组织培养对胞嘧啶甲基化的影响,利用MSAP技术研究了随机选取的普通黄瓜‘长春密刺’实生苗与组织培养再生苗之间胞嘧啶甲基化差异,结果发现在胞嘧啶甲基化水平上,实生苗与组织培养再生苗之间没有差异。对甲基化模式进行进一步研究分析发现,与实生苗相比,组培再生苗部分位点发生了甲基化模式的变化,但变化位点仅占总位点的0.6%。暗示了组织培养不能引起胞嘧啶甲基化发生很大程度的变化。更多还原

【Abstract】 Polyploidy is a prominent process in higher plants and has played an important role in the evolutionary history of plants.Recent studies indicate that extensive genetic and epigenetic changes often occur at the early stage of allopolyploid’s genome formation,these changes have played a significant role in stabilization of newly formed genome of species. Though,many investigations were focused on the characters and reasons for allopolyploid induced genomic changes,the mechanisms remain elusive,because of the lack of information during allopolyploid evolution.Although,the development of molecular markers and comparative genomics have provide important basis for understanding of hybridization and allopolyploidization,the mechanisms still remain poorly understood, because of the complicated molecular evolutionary mechanism.Synthesized allopolyploids can be a very useful model system to study polyploidization, as the information at the early stage can be obtained exactly.With the newly formed allopolyploids,the genomic changes can be studied accurately compared to their diploid parents,which might help enriching the evolutionary theory.In this work,the chromosome behavior,genomic changes and DNA methylation pattem changes of a newly formed Cucumis amphidiploid were studied,in order to investigate the mechanisms of genetic and epigenetic changes.The detailed results as follows:1.Meiotic chromosome behavior and pollen fertility of the interspecific hybrid F₁ and amphidiploidThe meiotic chromosome behavior and pollen fertility of the F₁ hybrid（2n = 2X = 19） and self-cross progenies(S_1-4) of amphidiploid（2n = 4X = 38） from Cucumis hystrix chakr（2n = 2X = 24）×C.sativus L（2n = 2X = 14） were studied.The results showed that at metaphase I（MI）,the chromosome configuration of F₁ hybrid was 16.75Ⅰ+0.5Ⅱ+0.25Ⅲ+0.13Ⅳ.Most chromosomes in the F₁ were univalent,while in the amphidiploid were bivalent.At metaphaseⅡ,the F₁ could not produce normal tetrads but mostly polyads,while the amphidiploid mainly produced tetrads.Moreover,the frequency of bivalent at MI,tetrads at anphaseⅡand fertility of pollen increased during the self-cross course that indicated the improved cytogenetic stability of the amphidiploid.It was also found that at anaphase I（AI） and AⅡthe PMCs contained lagging chromosome,bridge fragment,unequal disjunctions and nonsynchronized disjunctions in amphidiploid,which might cause the low pollen fertility.2.Studies on sequence changes in the early genomes of interspecific F₁ hybrid and amphidiploid in CucumisIn the present study,extensive genomic changes were detected in newly obtained allotetraploid of Cucumis,namely Cucumis hytivus Chen and Kirkbride（C.hytivus）,by AFLP analysis using 12 pairs of EcoR-I/Mse-I primers.The results indicated that extensive genomic changes occurred at the early stage of allopolyploid genome.The genomic changes occurred form F₁ and the greatest changes of sequence was occurred from S₀ generation to S₁ generation,while,there were miner changes from S₁ to S₂, which indicated that the allopolyploid genome became more stable following the selfing process and the sequence change was a rapid event.Most genomic changes involved loss of parental restriction fragments and appearance of novel fragments,with some fragments disappearing in one generation and reappearing in others.Further investigation suggested that most of the changed sequences were related to repetitive sequence.In addition,it was observed that some variable bands were inherited preferentially from the male parent.During diploidization,sequence loss in reciprocal alloployploids often appears variable in different species,suggesting that the nuclear-cytoplasmic interactions play different roles in different polyploid plants.In this study,Amplified fragment length polymorphism（AFLP） analysis was carried out by using 23 pairs of EcoRI-NN/MseI-NNN selective primers in amplification of the DNAs from reciprocal F₁ hybrids of Cucumis hystrix Chakr.and C. sativus L.,the synthetic amphidiploids and the diploid parents.The results indicated that extensive DNA sequence loss was occurred in the reciprocal F₁ hybrids and amphidiploids. The frequency of loss of parental sequences was not statistically significant between the reciprocal crosses;moreover,the time（both started in the F₁） and the type of loss were also the same,suggesting that the nuclear-cytoplasmic interactions might not be the main factor causing sequence loss.In addition,the frequency of lost sequence from the C.sativus genome was more than from the C.hystrix,indicating that the sequence loss was more common from the parent,which has fewer chromosomes.3.Changes of cytosine methylation induced by wide hybridization and allopolyploidy in CucumisTo explore the molecular involvement of epigenetic phenomena,cytosine methylation of interspecific F₁ hybrids,allotetraploid and parental genome was investigated by using methylation-sensitive amplified polymorphism in Cucumis.Results from analysis of the methylation pattern found that methylation pattern changed in both the F₁ hybrids and in the allotetraploid genome compared to their corresponding parents,suggesting that wide hybridization and allopolyploidization induced cytosine methylation changes.Furthermore, these methylation changes could be classfied into five types according to the changed pattern,mainly including hyper-methylation and de-methylation,moreover,the hyper methylated sites was 2 to 4 fold more than the de-methylated sites,indicating that most of the changed sites showed an increase in cytosine methylation.The results also found that a majority of the methylated sites were from the maternal parent,which suggested that DNA methylation have played a significant role in protecting the maternal genome.Observations in different generations of the allopolyploids found that the extent of cytosine methylation pattern changes between the S₁ and S₂ were significantly higher than that between the S₂ and S₃,suggesting stability in advanced generations which further supported the hypothesis that DNA methylation plays a significant role in the diploidization.Analysis of the methylaion sensitive sequences induced by wide hybridization and allopolyploidization indicated that among 15 cloned sequences,seven of them showed high similarity to the known functional genes or genes involved in regulating gene expression. Specific primers were designed and the results from PCR confirmed that the polymorphisms were from the DNA modification.In addition,the reverse transcription-polymerase chain reaction analysis suggested that cytosine methylation might be related to regulation of gene expression,which further supported the hypothesis that DNA methylation plays a significant role in the allopolyploidization.Southern blot analysis indicated that the altered sequences referred to both single-copy and multi-copy sequence.Plantlets and seedlings of randomly selected common cucumber ’Changchun mici’ were studied to investigate the cytosine methylation changes induced by tissue culture using methylation-sensitive amplified polymorphism.Results revealed that there were no significant differences in the level of cytosine methylation in the plantlets and seedlings. Analysis of the methylation pattern found that only 0.6%of total sites changed in plantlets compared to the seedlings,suggesting that tissue culture could not induce a large array of cytosine methylation changes.更多还原